Neutralizing antibodies in milk and blood of lactating women vaccinated for SARS-CoV-2: a systematic review

ABSTRACT Objective: To compare the presence of neutralizing antibodies against SARS-CoV-2 found in the breast milk and blood of vaccinated lactating women with those not vaccinated. Data source: The study was registered in the International Prospective Register of Systematic Reviews (PROSPERO) under CRD42021287554 and followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Cohort, case-control, and cross-sectional studies that evaluated antibodies against SARS-CoV-2 in the milk and blood of vaccinated mothers and had as control group unvaccinated mothers were eligible. Health Sciences Descriptors (DeCs), Medical Subject Headings (MeSH) and Emtree descriptors were used for the Virtual Health Library (VHL), Medical Literature Analysis and Retrieval System Online (Medline/Pubmed), and Embase databases, respectively. In the Web of Science and Scopus, the strategy was adapted. No restrictions on the publication period and language were set. Data synthesis: The search identified 233 records, of which 128 duplicates and 101 papers that did not meet the inclusion criteria were excluded. Hence, four cohort studies were eligible. Nursing mothers vaccinated with the Pfizer-BioNTech and Moderna vaccines showed antibodies against SARS-CoV-2 in their blood and breast milk. Conclusions: Vaccinated lactating women had higher levels of immunoglobulin G (IgG) and A (IgA) in serum and breast milk than unvaccinated women.


INTRODUCTION
The unbridled spread of COVID-19 cases mobilized scientists around the world to develop vaccines with different technologies to combat the virus.The first immunizers available in the United States (Pfizer-BioNTech and Moderna) were messenger RNA (mRNA).Some vaccines ( Janssen-Johnson & Johnson, AstraZeneca, Sputnik-V, and CanSino) were developed using human and primate adenovirus vectors.The vaccines from Bharat Biotech, Sinopharm, and Sinovac used the entire inactivated SARS-CoV-2 virus as technology. 1 As expected, pregnant women were excluded from clinical trials for the development of vaccines against COVID-19. 2However, the American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal-Fetal Medicine (SMFM) recommended that all pregnant, postpartum, and breastfeeding women be vaccinated against the SARS-CoV-2 virus. 3ome studies have found that vaccination against COVID-19 induces an effective neutralizing antibody titer response in blood and breast milk. 4,5Romero Ramírez et al., 5 in a prospective cohort study with a convenience sample of 122 participants (98 vaccinated during lactation and 24 controls), observed that all vaccinated lactating women presented with anti-SARS-CoV-2 antibodies in serum and milk samples.Guida et al., 4 in a prospective cohort study, collected milk and serum samples from ten lactating women immunized with the mRNAB-NT162b2 vaccine.The authors detected anti-SARS-CoV-2 antibodies 20 days after application of the first dose of the vaccine in all serum samples; however, only in two milk samples, they detected a low level of antibodies.They also found the presence of anti-SARS-CoV-2 antibodies in all serum and milk samples seven days after the application of the second dose of the vaccine.
Nevertheless, it is crucial to emphasize that studies that reviewed the presence of antibodies against the new coronavirus in breast milk after vaccination are scarce, especially in Brazil.[10] Given this, there was a need to elucidate the expression of antibodies in the milk and blood of breastfeeding women vaccinated against the new coronavirus.Therefore, this study aimed to compare the presence of neutralizing antibodies against SARS-CoV-2 found in the breast milk and blood of vaccinated lactating women with those women who were not vaccinated.

METHOD
A systematic review was undertaken and registered in the Prospective Register of Systematic Reviews (PROSPERO) under CRD42021287554.The study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines 11 detailed in Figure 1.
From the definition of the key question, a systematized search strategy was developed using the Health Sciences Descriptors (DeCS) in the information sources available in the Virtual Health Library (VHL), the Medical Subject Headings (MeSH) indexing articles in Medical Literature Analysis and Retrieval System Online (Medline/Pubmed), and Emtree descriptors for searching in Embase.For the multidisciplinary databases Web of Science and Scopus, the strategy wording was adapted.
Database searches were carried out in July 2023 by two independent researchers in order to identify all studies that contained the desired keywords, including all combinations and free terms of the words: COVID-19, human milk, vaccine, and antibodies, and their Portuguese counterparts.The descriptors were combined with each other through the Boolean connector OR, within each set of terms, and then crossed with the Boolean connector AND.No restrictions on publication period and language were set.The search strategy is available with the corresponding author.
Observational cohort, case-control, or cross-sectional studies that assessed the presence of neutralizing antibodies against SARS-CoV-2 in the milk and blood of vaccinated breastfeeding women as well as unvaccinated breastfeeding women (control group) were qualified as eligible.Case reports, systematic reviews with or without meta-analysis, articles that analyzed antibodies in infected or non-lactating women, and studies that did not provide comparison outcome data between groups that received or did not receive the vaccine were excluded.
The search strategy outlined above was used to obtain titles, abstracts and full texts of studies likely to be relevant for the review.Two reviewers manually and independently screened against predefined inclusion criteria to identify studies regarding the presence of neutralizing antibodies in the milk and blood of nursing mothers vaccinated against COVID-19.Review conflicts were settled by a third reviewer.
The risk of bias among the articles included was also determined independently by the same reviewers based on the Newcastle-Ottawa Scale (2021) assessment tool.This scale contains eight items in three domains: group selection (0-4 points), goodness of fit for confounding (0-2 points), and assessment of exposure after outcome (0-3 points).The score for this tool 3 Rev Paul Pediatr. 2025;43:e2023210 RPPed varies from 7 to 9, 4 to 6, and 0 to 3, being considered high, moderate, and low quality, respectively. 12ata from the selected articles were tabulated according to the following items: name of authors, year, country, study design, vaccine technology and commercial name, follow-up time, sample size of vaccinated breastfeeding women, sample size of unvaccinated breastfeeding women, eligibility criteria, exclusion criteria, types of antibodies found in the blood and milk of the vaccinated and unvaccinated group, limitations, and main results.
Due to the heterogeneity between studies, a meta-analysis of the papers included in our survey was not feasible.However, the results were systematically summarized and reported.

RESULTS
Database searches identified 233 records, of which 128 duplicates were removed.Titles and abstracts of the remaining 105 papers were read and 94 were excluded.After reading 11 articles in full, four studies were considered eligible and, therefore, included in this systematic review.The study selection process was described in the PRISMA flowchart (Figure 1).All selected papers were cohort studies and had a follow-up period of postpartum women between 43 and 121 days.The four studies included were conducted in Spain, Israel, the United States of America, and Poland, and were published in 2021.Details of the studies and population characteristics are presented in Table 1.
There was variability in the sample size of the selected studies ranging between 22 and 124 women.The mean/median maternal age was greater than 30 years.All breastfeeding women received two doses of immunization against the SARS-CoV-2 virus using mRNA technology vaccines (Pfizer-BioNTech or Moderna), as recommended by each manufacturer.In none of the studies, vaccine booster doses were given to lactating women.
The evaluation of the quantity and type of antibodies found in the milk and blood of lactating women after  RPPed vaccination was carried out differently in all four studies.Jakuszko et al. 13 performed the evaluation four times, Valcarce et al. 14 twice, while Nir et al. 15 and Romero Ramírez et al. 5 just once.The antibodies evaluated and the main outcomes are presented in Table 2.

Identification of studies through databases and registries
Based on the immune response to vaccination against the SARS-CoV-2 virus among vaccinated compared to unvaccinated breastfeeding women, it was found that after vaccination with the Pfizer-BioNTech or Moderna immunizers, all blood and breast milk samples from the immunized women were positive for IgG antibodies.][15] It was also found that vaccinated breastfeeding women had higher levels of IgA in serum and milk than those who did not receive immunization and had an increase in IgA results after a second dose. 13,14nly two studies analyzed immunoglobulin M (IgM) in samples.Jakuszko et al. 13 found that this antibody was undetectable in all samples.Romero Ramírez et al. 5 evaluated IgM only in breast milk and observed an increase in its quantity among vaccinated breastfeeding women.
All selected articles had a score of eight points indicating high methodological quality performance (Table 3).

DISCUSSION
Until the registration date in PROSPERO, this is the first systematic review that compared the antibodies found in the blood and human milk of breastfeeding women vaccinated against the SARS-CoV-2 virus with those not vaccinated.
Vaccination works as the entry of non-pathogenic antigens in the blood that interact with the immune system in charge of the body's defense 16 and causes the production of antibodies which are glycoprotein molecules capable of detecting, recognizing, and eliminating the antigen. 17Antibodies have five basic forms called immunoglobulins (IgG, IgA, IgM, IgD, and Table 1.Characteristics of selected studies on the effect of vaccines on the presence of neutralizing antibodies found in the milk and blood of lactating women immunized against SARS-COV-2, 2023.mRNA: messenger RNA; HIV: human immunodeficiency virus; NI: not informed.*The group of vaccinated lactating women is the same group of unvaccinated (i.e., unexposed) women; however, before immunization.Table 3. Newcastle-Ottawa Scale quality assessment of each cohort study included. 12kuszko et al. 13 Nir et al. 15 Romero Ramírez et al. 5 Valcarce et al.RPPed IgE) 18 that can be found in various body fluids such as saliva, gastrointestinal fluid, blood, or breast milk. 19reast milk offered to newborns is extremely rich in both nutritional composition and non-nutritive bioactive factors, such as antibodies that contribute to reducing infant morbidity and mortality through immunological protection as well as reducing contamination risk. 20,21he World Health Organization (WHO) recommends exclusive breastfeeding until six months of age and the continuation of breastfeeding combined with complementary foods for two years or more. 22Even considering the finding of SARS-CoV-2 viral particles in human milk, breastfeeding was recommended among women with suspected or confirmed disease given that viral transmission through human milk, if any, is quite rare 23 and that its benefits for both women and newborns significantly outweigh the risk of transmitting this virus to newborns. 244][15] Furthermore, an increase in the number of antibodies was observed after the administration of a second dose of the vaccine, which confirms the importance of completing the vaccination schedule as recommended by the manufacturers. 13,14egarding the types of immunoglobulins evaluated in the studies, it was observed that the IgM was not measured by Nir et al. 15 and Valcarce et al. 14 .Jakuszko et al. 13 did not detect this immunoglobulin in the milk and blood samples from lactating women (including those samples collected after vaccination).Only Romero Ramírez et al. 5 detected IgM in breast milk samples, albeit in minimal quantities.This result was possibly due to IgM being found mainly when the infection is still active; it generally peaks in the first week after infection and disappears from the peripheral blood earlier than IgG. 25,26gG was detected in the milk and blood of vaccinated lactating women who made up the sample of the four selected studies, 5,[13][14][15] while among those not vaccinated, IgG was minimally detected in two studies.5,14 It is interesting that IgG is generally the most abundant antibody in serum and is mainly synthesized in the secondary immune response to pathogens.IgG can activate the classical pathway of the complement system and is also highly protective.27 As for IgA, Jakuszko et al. 13 and Valcarce et al. 14 observed that vaccinated breastfeeding women had higher levels of IgA in serum and milk than unvaccinated women.Romero Ramírez et al. 5 found that only human milk samples were positive for IgA in both groups.Nir et al. 15 in turn, did not analyze the presence of IgA in any of the samples.IgA is the second most abundant immunoglobulin after IgG; it is present in external secretions such as saliva, tears, colostrum, and respiratory and intestinal secretions.Therefore, IgA is important in the primary immunological line of defense against local infections in sites such as the respiratory and gastrointestinal tracts. 27 Effiient transfer of anti-SARS-CoV-2 IgG antibodies to newborns through the placenta of women vaccinated during pregnancy with BNT162b2 mRNA technology has been reported.15 Corroborating this result, Collier et al., 28 in a cohort study of pregnant women vaccinated against COVID-19 during pregnancy, also detected the presence of anti-SARS-CoV-2 IgG in maternal serum at birth and in the umbilical cord blood, suggesting the existence of efficient transplacental transfer of maternal antibodies.

Reference/ country of study
Other vaccines, such as influenza, also increased antibodies in the milk and maternal blood of vaccinated women.Schlaudecker et al., 29 in a prospective study conducted in Bangladesh, analyzed the breast milk and serum of 340 women who received the influenza vaccine.The results of this study showed that IgA anti-influenza antibodies were higher in the breast milk and blood of mothers who received the vaccine.Furthermore, the sustained high levels of specific IgA in breast milk from women immunized during pregnancy suggest that breastfeeding may provide vaccine-specific local mucosal protection for the infant up to six months of age.
Abu Raya et al., 30 aiming to examine the levels of IgA and IgG specific to pertussis in breast milk, recruited 37 women (25 vaccinated with Tdap and 12 unvaccinated) to form a cohort.It was found that antibody levels against pertussis in breast milk were significantly higher among women vaccinated with Tdap during pregnancy than among those not vaccinated.
In this connection, based on the selected studies, we could verify that the vaccine against COVID-19, like other vaccines approved for use in pregnant and postpartum women, such as those against influenza, tetanus, diphtheria, and pertussis, increases the number of antibodies in the milk and blood of women who chose to receive immunization against the SARS-CoV-2 virus. 29,30n short, three studies included in this review 5,13,14 found a highly significant correlation between the antibody levels in serum samples and breast milk.Thus, the presence of antibodies in the blood appears to predict the appearance of antibodies in human milk.
It is important to highlight that in order to confirm that mothers vaccinated against COVID-19 can transfer passive immunity to their newborns through breastfeeding, capable of potentially protecting them against SARS-CoV-2 infection, future studies are needed on the effects of vaccination on newborn antibodies and the neutralization capacity of these antibodies.
Another important aspect to be considered to elucidate the possible benefits of vaccination in maternal and child protection against the SARS-CoV-2 virus is what would be the ideal time to administer the vaccine (first, second, or third trimester of pregnancy, or even, during lactation).The study by Mithal et al., 31 carried out with women vaccinated against COVID-19 during pregnancy, demonstrated that the transfer of antibodies appears to increase with the latency of vaccination and suggested that, at least among women in the third trimester of pregnancy, early vaccination may produce greater neonatal immunity.However, Yang et al., 32 in a retrospective cohort study with 1,359 pregnant women, stated that immunization in any trimester of pregnancy, or before pregnancy, is associated with detectable levels of maternal anti-SARS-CoV-2 antibodies at delivery although the highest levels of maternal and consequently cord blood antibodies occur with vaccination performed early in the third trimester of pregnancy.
Since research on aspects of the SARS-CoV-2 virus is extremely important given the short period of discovery of the virus and simultaneously the importance of studying the effects of any and all immunizations, this systematic review provides an expanded view of the state of the art on the expression of maternal antibodies against COVID-19 in those vaccinated women, thus providing an assumption that vaccination is the most promising and safe way of maternal and child protection.
Regarding limitations, only two studies compared the number of antibodies between vaccinated and unvaccinated breastfeeding women; however, only Romero Ramírez et al. 5 and Jakuszko et al. 13 performed tests to gather evidence that those women were not infected or that they already had antibodies against COVID-19 in their blood at the time of collection.All studies reported the application of only mRNA-type vaccines; thus, we cannot generalize the results to other vaccine manufacturing technologies.Future studies should preferably evaluate the effect of other available vaccine technologies and the effect of booster doses of each of those vaccines.Valcarce et al. 14 considered the unexposed group (unvaccinated breastfeeding women) as an exposed group at a later date, after the breastfeeding women took the vaccine against the new coronavirus.Nir et al. 15 analyzed women who had recovered from COVID-19 with evidence of previous positive tests for SARS-CoV-2 as a control group.Furthermore, some significant differences in the studies included the participants' sample size, the collection methods, and the variability in the number of times the samples were analyzed.Finally, all studies included in this review were international, thus confirming the scarcity of studies on this topic in Brazil.In conclusion, it was found that after vaccination with the Pfizer-BioNTech or Moderna immunizers, all blood and breast milk samples from lactating women were positive for IgG antibodies, which were even more predominant in the blood.Likewise, vaccinated lactating women had higher levels of IgA in serum and milk than unvaccinated women.Furthermore, the immune response to vaccination against SARS-CoV-2 was improved after the administration of a second dose, that is, when breastfeeding women complete the vaccination schedule indicated by each vaccine manufacturer.Therefore, as breastfeeding women vaccinated against the SARS-CoV-2 virus showed higher antibody expression when compared with those not vaccinated, it is likely that, in addition to maternal protection against COVID-19, immunization also provides neonatal immunity through nursing.Thus, vaccination against COVID-19 in pregnant and postpartum women as well as nursing should be encouraged.
Records removed before screening: Duplicate records removed (n=128) Records marked as ineligible by automation tools (n=0) Records removed for other reasons (n=0) articles did not evaluate breast milk and blood simultaneously Selected records (n=105) Deleted reports: Population divergent from the objective of the study (pregnant or non-lactating women) (n=7) Reports assessed for eligibility (n=11) Studies included in the review (n=4) Included study reports (n=0) Included Screening Identification *Assessment of conflicts.Source: Page et al. 11

Figure 1 .
Figure 1.Flowchart of the article selection process. 11

Table 2 .
Characteristics of technologies used, antibodies evaluated, and main results.